Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt

The increasing rates of antimicrobial resistance among carbapenem-resistant Acinetobacter baumannii in the Middle East and North Africa are one of the major concerns for healthcare settings. We characterised the first A. baumannii isolate harbouring five β-lactamases identified in Egypt. The isolate Ale25 was obtained from an ICU patient of a hospital from Alexandria. The isolate was phenotypically and genotypically screened for carbapenemase genes. The isolate was resistant to carbapenems, aminoglycosides, fluoroquinolones and cefiderocol. Whole-Genome Sequencing identified five β-lactamase genes, blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57, together with other antibiotic resistance genes, conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol. Virulome analysis showed the presence of genes involved in adhesion and biofilm production, type II and VI secretion systems, exotoxins, etc. Multi-Locus Sequence Typing analysis identified the isolate as Sequence Types 113Pas and 2246Oxf, belonging to International Clone 7. Sequencing experiments revealed the presence of four plasmids of 2.7, 22.3, 70.4 and 240.8 Kb. All the β-lactamase genes were located in the chromosome, except the blaPER-7, gene which was found within the plasmid of 240.8 Kb. This study highlights the threat of the emergence and dissemination of these types of isolates.


Introduction
Carbapenem-resistant Acinetobacter baumannii (CRAB) constitutes one of the major challenges for healthcare settings due to its high rates of antimicrobial resistance, heading the list of critical pathogens published by The World Health Organization. The main mechanisms of carbapenem resistance in A. baumannii are OXA-type carbapenemases [1]. However, the number of A. baumanni isolates harbouring class B metallo-β-lactamases, such as New Delhi Metallo-Beta-lactamases (NDM), have dramatically increased [1]. In Table 1. Minimum Inhibitory Concentrations (MICs) and interpretations of the A. baumannii isolate Ale25. "R" and "S" corresponds to resistant and susceptible, respectively. "IE" indicates that there is insufficient evidence that the organism or group is a good target for therapy with the agent. Molecular typing assigned the isolate to STs 113 by Pasteur Scheme and ST 2246 by Oxford Scheme. These results in combination with the bla OXA-51-like variant revealed that the isolate belonged to IC7.

Antibiotic MIC (mg/L) Interpretation
Three recognised carbapenemase genes were detected by Whole Genome Sequencing, bla OXA-64 , bla NDM-1 and bla OXA-23 , as well as the Extended Spectrum Beta-Lactamase coding gene bla PER-7 and the cephalosporinase gene bla ADC-57 . The isolate also harboured several aminoglycoside resistance genes conferring resistance to streptomycin, gentamycin, tobramycin, amikacin, kanamycin and spectinomycin, as well as genes conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol, respectively ( Table 2). Analysis of the genetic surroundings of the β-lactamase genes located the bla NDM-1 within the truncated isoform of transposon Tn125 (∆Tn125) (Figure 1a). The bla PER-7 gene was found within a complex structure connecting the ISCR1 element and a class 1 integron with part of IS26 upstream of the integron. The gene-cassette variable regions contained the antibiotic resistance genes arr-2 and cmlA5 genes (Figure 1b). Upstream of bla PER-7 , we found the ISCR1 element. A gst gene and an abc transporter gene were detected in the ISCR1 linked genes variable region downstream of bla PER-7 . Interestingly, another ISCR1 element, IS5, and part of IS10A were found downstream of the 3 -CS. The bla OXA-23 gene was located within transposon Tn2006 (Figure 1c). No insertion sequences were found in close proximity to bla OXA-64 or bla ADC-57 genes (Figure 1d,e). aph (3′)-VI ant (3″)-IIa 1 Recognised carbapenemase genes.
Analysis of the genetic surroundings of the β-lactamase genes located the blaNDM-1 within the truncated isoform of transposon Tn125 (ΔTn125) (Figure 1a). The blaPER-7 gene was found within a complex structure connecting the ISCR1 element and a class 1 integron with part of IS26 upstream of the integron. The gene-casse e variable regions contained the antibiotic resistance genes arr-2 and cmlA5 genes (Figure 1b). Upstream of blaPER-7, we found the ISCR1 element. A gst gene and an abc transporter gene were detected in the ISCR1 linked genes variable region downstream of blaPER-7. Interestingly, another ISCR1 element, IS5, and part of IS10A were found downstream of the 3′-CS. The blaOXA-23 gene was located within transposon Tn2006 (Figure 1c). No insertion sequences were found in close proximity to blaOXA-64 or blaADC-57 genes (Figure 1d,e). Virulome analysis revealed the presence of a wide variety of genes involved in adherence and biofilm production (type IV pili, biofilm-controlling response regulator, Ade-FGH efflux pump, quorum sensing, Csu fimbriae, biofilm-associated protein, outer-membrane protein, Poly-N-acetyl-D-glucosamine), immune modulation (capsule, lipopolysaccharide and penicillin-binding Protein G), effector delivery systems (type II and type VI secretion systems), exotoxins (phospholipases C and D) and iron uptake (acinetobactin) ( Table 3). Virulome analysis revealed the presence of a wide variety of genes involved in adherence and biofilm production (type IV pili, biofilm-controlling response regulator, AdeFGH efflux pump, quorum sensing, Csu fimbriae, biofilm-associated protein, outer-membrane protein, Poly-N-acetyl-D-glucosamine), immune modulation (capsule, lipopolysaccharide and penicillin-binding Protein G), effector delivery systems (type II and type VI secretion systems), exotoxins (phospholipases C and D) and iron uptake (acinetobactin) ( Table 3). Table 3. Virulome of the A. baumannii isolate Ale25. Genes are represented in italics. BCRR-Biofilm-controlling response regulator; BAP-Biofilm-associated protein; OMP-Outer-membrane proteins; PNAG-Poly-N-acetyl-D-glucosamine; PBPG-Penicillin-Binding Protein G; LPS-Lipopolysaccharide; T2SS-Type II secretion system; T6SS-Type VI secretion system.  Figure 2A) and a plasmid of approximately 240 Kb ( Figure 2B) were observed by conventional plasmid extraction and S1-Pulsed Field Gel Electrophoresis experiments, respectively. Further sequencing experiments revealed three conjugative plasmids of 22.3, 70.4 and 240.8 Kb as well as a non-conjugative plasmid of 2.7 Kb. PCR-based and whole-genome-sequence-based Acinetobacter Replicon typing experiments identified repAci6 (homology group GR6) in the 70.4 Kb plasmid and a rep gene encoding a Rep_3 family protein R3-T60 in the 240.8 Kb plasmid. We were not able to identify the rep genes of the 2.7 and 22.3 Kb plasmids using these methods. three conjugative plasmids of 22.3, 70.4 and 240.8 Kb as well as a non-conjug of 2.7 Kb. PCR-based and whole-genome-sequence-based Acinetobacter Rep experiments identified repAci6 (homology group GR6) in the 70.4 Kb plasm gene encoding a Rep_3 family protein R3-T60 in the 240.8 Kb plasmid. We to identify the rep genes of the 2.7 and 22.3 Kb plasmids using these method Plasmid sequencing experiments located all the β-lactamase genes in some (Supplementary Materials Tables S1-S5), except the blaPER-7 gene, whic within the 240.8 Kb plasmid pAbAle25.1 together with aminoglycoside (arm sulfonamide (sul1, sul2), macrolide (msrE, mphE), tetracycline (tet(B)), chlo (cmlA5) and rifamycin (arr-2) resistance genes ( Figure 3). The aminoglycosi gene aph (3′)-VI was found within the 70.4 Kb plasmid. Hybridisation exp confirmed the chromosomal location of the blaNDM-1 gene ( Figure 2B).  Plasmid sequencing experiments located all the β-lactamase genes in the chromosome (Supplementary Materials Tables S1-S5), except the bla PER-7 gene, which was found within the 240.8 Kb plasmid pAbAle25.1 together with aminoglycoside (armA, strA, strB), sulfonamide (sul1, sul2), macrolide (msrE, mphE), tetracycline (tet(B)), chloramphenicol (cmlA5) and rifamycin (arr-2) resistance genes (Figure 3). The aminoglycoside resistance gene aph

Discussion
Consistent with the recent studies reporting a carbapenem resistance rate of 98% among Egyptian A. baumannii isolates [5], our isolate was resistant to imipenem and meropenem. To our knowledge, no cefiderocol-resistant A. baumannii isolates have been previously reported in Egypt, turning the spotlight on the emergence and dissemination of resistance to cefiderocol in Egypt.
The isolate belonged to IC7, which is especially interesting considering that isolates belonging to IC7 are frequently reported in South America [6,7] but not in Egypt, where IC2 is the most prevalent international clone [8].
The presence of ISAba125 upstream of the blaNDM-1 may enhance its expression [9]. In our isolate, blaNDM-1 gene was located in the chromosome, which is the most frequent localization of these genes, as previously reported by other authors [1,10]. By BLASTn, we observed that the genetic environment of the blaNDM-1 gene was 100% similar to an A. baumannii isolated in France in 2011 (Acc. No. JX000237.3), an A. baumannii isolated in

Discussion
Consistent with the recent studies reporting a carbapenem resistance rate of 98% among Egyptian A. baumannii isolates [5], our isolate was resistant to imipenem and meropenem. To our knowledge, no cefiderocol-resistant A. baumannii isolates have been previously reported in Egypt, turning the spotlight on the emergence and dissemination of resistance to cefiderocol in Egypt.
The isolate belonged to IC7, which is especially interesting considering that isolates belonging to IC7 are frequently reported in South America [6,7] but not in Egypt, where IC2 is the most prevalent international clone [8].
The presence of ISAba125 upstream of the bla NDM-1 may enhance its expression [9]. In our isolate, bla NDM-1 gene was located in the chromosome, which is the most frequent localization of these genes, as previously reported by other authors [1,10]. By BLASTn, we observed that the genetic environment of the bla NDM-1 gene was 100% similar to an A. baumannii isolated in France in 2011 (Acc. No. JX000237.3), an A. baumannii isolated in Lebanon in 2015 (Acc. No. CP082952.1) and an A. baumannii isolated in India in 2018 (Acc. No. CP038644) assigned to ST85 Pasteur Scheme, ST1089 Oxford Scheme with bla OXA-94 , and which harboured a single copy of bla NDM-1 . These three isolates belonged to IC9, suggesting that this genetic context is circulating and being transmitted among different ICs.
The structure containing the ISCR1 element and a class 1 integron, in which the bla PER-7 gene was located, has been previously described, and it is closely related to multidrugresistant bacteria [11]. The presence of this betalactamase gene in a high-molecular-weight conjugative plasmid of 240.8 Kb has not been previously reported, although the bla PER-1 variant has been described by other authors within a similar structure [12]. Our concern is that it is the first time one obtains the sequence of this structure coding for multiple resistance and virulence genes. Despite the fact that the carbapenemase activity of PER-type and ADC-like enzymes is doubtful, recent studies have shown that PER-7 may exhibit carbapenemase activity [9] and that ADC-57 may hydrolyse ertapenem [13], which, in our isolate, may be contributing to carbapenem resistance.
The bla OXA-23 gene was found within the transposon Tn2006, which is the most frequently reported transposon harbouring this gene and the only one that has been experimentally proven to transpose [14,15]. The presence of ISAba1 upstream of the bla OXA-23 gene might lead to the overexpression and mobilisation of this carbapenemase gene, enhancing carbapenem resistance [16].
The results we obtained by different plasmid extraction methods showed that in order to obtain the complete profile, a combination of all of them is needed, as this improves the number of structures that can be detected. Each technique is not able to provide a complete landscape of the plasmidome that was finally revealed by sequencing experiments. Plasmid extraction kits and conventional lysis are useful for low-molecular-weight plasmids isolation and for a first screening, but it becomes necessary to complete these results with additional experiments, such as S1-PFGE or sequencing techniques. The combination of using short and long-read technologies to perform hybrid assemblies has been demonstrated to be a powerful tool for plasmids resolution [17].
To the best of our knowledge, only a single isolate harbouring four different classes of β-lactamase genes has been previously reported in Bangladesh [18], so this would be the first report of an A. baumannii clinical isolate harbouring four different classes of βlactamase genes in Egypt, and it would also be the first cefiderocol-resistant isolate reported in Egypt. This study highlights the ability of A. baumannii to acquire and accumulate multiple antibiotic resistance genes, and it also turns the spotlight on the emergence and threat of the dissemination of IC7 isolates, not frequently reported in Egypt, and the importance of controlling the dissemination of these types of isolates.

Materials and Methods
A 63-year-old renal dialysis female patient with acute-on-chronic kidney disease was admitted to the hospital and entered the ICU on 13 November 2020. On 29 November 2020, a swab from around the abdominal pigtail catheter was obtained by the Microbiology Service, Alexandria University Medical Research Institute. Being a renal patient, she was treated with tigecycline injection as well as topical polymyxin. The patient was discharged home after 40 days at the ICU.
To investigate the presence of plasmids, we first performed plasmid extractions using GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer's indications, and S1-Pulsed-Field Gel Electrophoresis (PFGE) experiments. The Bacterial DNA embedded in agarose plugs was digested using 14 units of S1-nuclease (Takara Bio, Kusatsu, Japan) per plug, and then it was run on a CHEF-DR III system (Bio-Rad, Munich, Germany) for 18 h at 6 V/cm and 14 • C. CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as a molecular weight marker. Southern-blot hybridisation of the resulting PFGE was performed to locate bla NDM-1 gene with specific digoxigenin-labelled DNA probes (Roche, Mannheim, Germany). Signal detection was performed using DIG Nucleic Acid Detection Kit (Roche).

Data Availability Statement:
The assembled genome is available in NCBI under the accession number JANBZS000000000.

Conflicts of Interest:
The authors declare no conflict of interest.